Differential interactions of G-proteins and adenylyl cyclase with nucleoside 5′-triphosphates, nucleoside 5′-[γ-thio]triphosphates and nucleoside 5′-[β,γ-imido]triphosphates

Gille, Andreas; Guo, Zhixiong; Mou, Tung-Chung; Doughty, Michael B.; Lushington, Gerald H.; Seifert, Roland

doi:10.1016/j.bcp.2005.10.006

Cited by 18 publications

(33 citation statements)

References 32 publications

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“…However, it should be emphasized that the inhibitory effect of metoprolol on GTP-stimulated AC activity did not reach statistical significance, indicating that the constitutive activity of the ␤ 1 -adrenoceptor at least in this system is small. The inhibitory effect of GTP Ͼ10 M on AC activity is due to competition of GTP with ATP at the catalytic site of AC (Gille et al, 2005). The stimulatory effect of isoproterenol on AC in the absence of added GTP is explained by nucleoside diphosphokinase-mediated conversion of endogenous GDP to GTP by ATP (Kimura and Shimada, 1988).…”

Section: Detection Of Ac Isoforms In Mousementioning

confidence: 99%

“…Inhibition curves were analyzed by nonlinear regression. (Gille et al, 2004(Gille et al, , 2005Taha et al, 2009). For comparison with previous studies, we conducted some AC studies with heart membranes both in the presence of Mg 2ϩ and Mn 2ϩ .…”

Section: Real-time Pcr and Immunoblot Studiesmentioning

confidence: 99%

“…Specifically, the use of the above-described buffers was critical. When hearts were homogenized in the standard buffer used for Sf9 cell homogenization (Houston et al, 2002;Gille et al, 2005), no AC activity was recovered. The reason for the striking impact of buffer composition on AC activity yield in mouse heart membranes is unknown.…”

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confidence: 99%

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Characterization of Mouse Heart Adenylyl Cyclase

Göttle

Geduhn²,

König³

et al. 2009

J Pharmacol Exp Ther

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Chronic heart failure is one of the most frequent causes of death in humans. Knockout of type 5 adenylyl cyclase (AC) in mice causes longevity and protection from cardiomyopathy, and an AC5 inhibitor reduces ␤-adrenoceptor-stimulated Ca 2ϩ inward currents in isolated mouse cardiomyocytes. These data indicate that selective AC5 inhibitors may be beneficial in chronic heart failure. Therefore, we characterized AC in mouse heart membranes. Real-time polymerase chain reaction and immunoblot analysis suggested that AC5 is an important heart AC isoform. Enzyme kinetics of heart AC and recombinant AC5 in the presence of Mg 2ϩ were similar. Moreover, the inhibitory profile of eight 2Ј(3Ј)-O-(N-methylanthraniloyl) (MANT)-nucleoside 5Ј-([␥-thio])triphosphates on mouse heart in the presence of Mg 2ϩ was almost identical to that of AC5. MANT-ITP was the most potent inhibitor of heart AC and recombinant AC5, with K i values in the 15 to 25 nM range in the presence of Mg 2ϩ and in the 1 to 5 nM range in the presence of Mn 2ϩ . However, in the presence of Mn 2ϩ , we also noted differences between mouse heart AC and AC5 with respect to enzyme kinetics and forskolin analog effects. In conclusion, with regard to expression and kinetics and inhibition by MANT-nucleotides in the presence of Mg 2ϩ , AC5 is an important AC isoform in heart, with MANT-ITP being an excellent starting point for the design of AC5-selective inhibitors. Unfortunately, a limitation of our study is the fact that immunologically and biochemically, AC5 and AC6 are quite similar, although they have different roles in heart. Moreover, lack of antibody specificity and Mn 2ϩ masking AC5 effects were problems.

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Section: Detection Of Ac Isoforms In Mousementioning

confidence: 99%

Section: Real-time Pcr and Immunoblot Studiesmentioning

confidence: 99%

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Characterization of Mouse Heart Adenylyl Cyclase

Göttle

Geduhn²,

König³

et al. 2009

J Pharmacol Exp Ther

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“…A recent report raised the question whether G proteins have to be in the GTP-bound form to be active (Ugur et al 2005;Wieland and Michel 2005), and there is increasing evidence that not all heterotrimeric G proteins dissociate into Gα and Gβγ upon activation (Bünemann et al 2003;Frank et al 2005). Taking further into account that, in in vitro systems, even nucleotide-free Gα subunits are active (Lutz et al 2002), or heterotrimeric G proteins can be activated by pyrimidine nucleotide triphosphates under conditions excluding high energy phosphate relay (Gille et al 2005), it is evident that investigations on heterotrimeric G proteins and their interaction partners, like those performed by Karl H. Jakobs since 1970 (Jakobs and Schultz 1970), will provide interesting and relevant topics for several decades of future research.…”

Section: Resultsmentioning

confidence: 99%

Interaction of nucleoside diphosphate kinase B with heterotrimeric G protein βγ dimers: consequences on G protein activation and stability

Wieland

2007

Naunyn-Schmied Arch Pharmacol

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“…Second, the selectivity of MANT-ITP for AC5 relative to other AC isoforms is only very moderate , because the catalytic site of the membranous AC isoforms is highly conserved . Third, one should also keep in mind that hypoxanthine-based nucleotides bind not only to mACs but also to other signaltransducing proteins, including soluble guanylyl cyclase and G-proteins (Seifert et al, 1999;Gille et al, , 2005Hü bner et al, 2011). Accordingly, pleiotropic effects unrelated to direct mAC inhibition could arise (Hü bner et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

Structural Basis for the High-Affinity Inhibition of Mammalian Membranous Adenylyl Cyclase by 2′,3′-O-(N-Methylanthraniloyl)-Inosine 5′-Triphosphate

Hübner¹,

Dixit²,

Mou³

et al. 2011

Mol Pharmacol

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2Ј,3Ј-O-(N-Methylanthraniloyl)-ITP (MANT-ITP)is the most potent inhibitor of mammalian membranous adenylyl cyclase (mAC) 5 (AC5, K i , 1 nM) yet discovered and surpasses the potency of MANT-GTP by 55-fold (J Pharmacol Exp Ther 329: 1156 -1165, 2009). AC5 inhibitors may be valuable drugs for treatment of heart failure. The aim of this study was to elucidate the structural basis for the high-affinity inhibition of mAC by MANT-ITP. MANT-ITP was a considerably more potent inhibitor of the purified catalytic domains VC1 and IIC2 of mAC than MANT-GTP (K i , 0.7 versus 18 nM). Moreover, there was considerably more efficient fluorescence resonance energy transfer between Trp1020 of IIC2 and the MANT group of MANT-ITP compared with MANT-GTP, indicating optimal interaction of the MANT group of MANT-ITP with the hydrophobic pocket.The crystal structure of MANT-ITP in complex with the G s ␣-and forskolin-activated catalytic domains VC1:IIC2 compared with the existing MANT-GTP crystal structure revealed only subtle differences in binding mode. The higher affinity of MANT-ITP to mAC compared with MANT-GTP is probably due to fewer stereochemical constraints upon the nucleotide base in the purine binding pocket, allowing a stronger interaction with the hydrophobic regions of IIC2 domain, as assessed by fluorescence spectroscopy. Stronger interaction is also achieved in the phosphate-binding site. The triphosphate group of MANT-ITP exhibits better metal coordination than the triphosphate group of MANT-GTP, as confirmed by molecular dynamics simulations. Collectively, the subtle differences in ligand structure have profound effects on affinity for mAC.

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Differential interactions of G-proteins and adenylyl cyclase with nucleoside 5′-triphosphates, nucleoside 5′-[γ-thio]triphosphates and nucleoside 5′-[β,γ-imido]triphosphates

Cited by 18 publications

References 32 publications

Characterization of Mouse Heart Adenylyl Cyclase

Characterization of Mouse Heart Adenylyl Cyclase

Interaction of nucleoside diphosphate kinase B with heterotrimeric G protein βγ dimers: consequences on G protein activation and stability

Structural Basis for the High-Affinity Inhibition of Mammalian Membranous Adenylyl Cyclase by 2′,3′-O-(N-Methylanthraniloyl)-Inosine 5′-Triphosphate

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